1. Field of the Invention
The present invention relates generally to the fields of pharmaceutical sciences, protein chemistry, polymer chemistry, colloid chemistry, immunology, and biomedical engineering. More specifically, the present invention relates to a novel microparticulate and nanoparticulate system for drug and antigen delivery, for gene (plasmid DNA) delivery and antisense RNA and DNA oligonucleotide delivery.
2. Description of the Related Art
Microparticulate systems are solid particles having a diameter of 1-2,000 xcexcm (2 mm) and more preferably 1-10 xcexcm (microparticles). Nanoparticulate system are submicroscopic colloidal particles (nanoparticles) having a diameter of preferably 50-500 nm (1,000 nm=1 xcexcm). Both microparticles and nanoparticles can be formed from variety of materials, including synthetic polymers and biopolymers (proteins and polysaccharides). Both microparticles and nanoparticles are used as carriers for drugs and other biotechnology products, such as antigens, genes and antisense oligonucleotides.
In the controlled drug and antigen delivery area, microparticles and nanoparticles are formed in a mixture with molecules to be encapsulated within the particles, for subsequent sustained release. A number of different techniques are routinely used to make these particles from synthetic or natural polymers, including phase separation, precipitation, solvent evaporation, emulsification, and spray drying, or a combination of thereof [Desay, P.B., Microencapsulation of drugs by pan and air suspension technique. Crit. Rev. Therapeut. Drug Carrier Syst., 5: 99-139 (1988); Berthold, A., Cremer, K., Kreuter, J. Preparation and characterization of chitosan microspheres as drug carrier for prednisolone sodium phosphate as model anti-inflammatory drugs. J. Controlled Release 39: 17-25 (1996); Watts, P. J., Davies, H. C., Melia, C. D. Microencapsulation using emulsification/solvent evaporation: An overview of techniques and applications. Crit. Rev. Therapeut. Drug Carrier Syst. 7: 235-159 (1990); Cowsar, D. R., Tice, T. R., Gilley, R. M., English, J. P. Poly(lactide-co-glycolide) microcapsules for controlled release of steroids. Methods Enzymol. 112: 101-116 (1985); Genta, I., 5 Pavanetto, F., Conti, B., Ginnoledi, P., Conte, U. Spray-drying for the preparation of chitosan microspheres. Proc. Int. Symp. Controlled Release Mater. 21: 616-617 (1994)].
Microparticles and nanoparticles can be prepared either from preformed polymers, such as polylactic acid, polylactic-glycolic acid [Cohen, et al., Controlled delivery systems for proteins based on poly(lactic/glycolic acid) microspheres. Pharm. Res. 8: 713-720 (1991)], or from a monomer during its polymerization, as is the case of polyalkylcyanoacrylates [Al-Khouri-Fallouh, et al., Development of new process for the manufacture of polyisobutylcyanoacrylate nanoparticles. Int. J. Pharm. 28: 125-132 (1986)]. Both of the above technologies have limited application because of the use of organic solvents during their preparation which leave residual organic solvents in the final product. Although the polyalkylcyanoacrylate nanoparticulate technology is also available as a water-based [Couvreur, et al., Biodegradable submicroscopic particles containing a biologically active substance and compositions containing them, U.S. Pat. No. 4,329,332 (1982)], animal studies demonstrated a presence of toxic degradation products [Cruz, et al., Interaction between polyalkylcyanoacrylate nanoparticles and peritoneal macrophages: MTT metabolism, NBT reduction, and NO production. Pharm. Res. 14: 73-79 (1997)].
Cell encapsulation [Chang, T. M. Hybrid artificial cells:
Microencapsulation of living cells. ASAIO Journal 38: 128-130 (1992)] is a related technology which has been also explored for the purpose of making microparticles and nanoparticles. Such particles can be formed either by polymer precipitation, following the addition of a non-solvent or by gelling, following the addition of a small inorganic ion (salt) and of a complexing polymer (of an opposite charge). If enough time is allowed, the particle interior (core) can be completely gelled. Usually, the inner core material is typically of a polyanionic nature (negatively charged polymer), the particle membrane (corona) is made from a combination of polycation (positively charged polymer) and polyanion. The core material is usually atomized (nebulized) into small droplets and collected in a receiving bath containing a polycationic polymer solution. The reverse situation is also possible in which case the core material is polycationic and the receiving bath is polyanionic.
Several binary polymeric encapsulation systems have been described. An undesirable side effect of these encapsulation systems is that the membrane parameters are tied by a single chemical complex resulting from the ionic interactions. The inability to adjust particle parameters independently has limited the success of this system.
To overcome these limitations, a new multicomponent polymeric particle was designed which allows for independent modification of mechanical strength and permeability. Over one thousand combinations of polyanions and polycations were examined as polymer candidates suitable for encapsulation of living cells. Thirty-three combinations were found to be usable. However, the composition and concentrations do not allow for generating of small microparticles and nanoparticles, suitable for drug and antigen delivery.
The prior art is deficient in the lack of effective means of drug and antigen delivery, as well as plasmid and oligonucleotide delivery. The present invention fulfills this longstanding need and desire in the art.
To overcome the limitations of the prior art, the present invention provides new combinations of multicomponent water-soluble polymers which allow for modification of the particle size down to a desirable size, adequate mechanical strength, and desirable permeability and surface characteristics.
In one aspect, the present invention provides a method of preparing of microparticles and nanoparticles by means of a hollow ultrasonic device and a combination of polymers at relatively low concentrations. In another aspect, the present invention provides a method of production of micro- and nanoparticles in a single step process. In another aspect, the present invention provides a composition of matter of micro- and nanoparticles whereas the multicomponent combination of polymers is composed of a structural (gelling) polymer and a polymer providing the mechanical strength (crosslinking) and permeability control. In another aspect, the present invention provides a composition of matter and method of incorporation of antigens as an integral part of the particulate matter. In another aspect, the present invention provides a composition of matter and method of electrostatic and steric stabilization of particles whereas the stabilizing polymers are integral part of the particulate matter. Also provided is a method of direct use of particles generated in the single step as a vaccine delivered orally, nasally, rectally or vaginally, through inhalation to the lung, and by injection into muscle or skin or underneath the skin.
In another aspect, the present invention provides a composition of matter and method of production whereas the particles comprise of anionic DNA or oligonucleotide incorporated as an integral part of the matter. Further provided is a method of post-production processing of particles, composed of recovery and washing steps.
In another embodiment, the present invention provides a method of stabilization and composition of matter of particles by means of physiological crosslinking agents. Also provided is a method of cryoprotection and stabilization by means of lyophilization. In addition, the present invention provides methods of adjustment of biodegradation and composition of matter of particles by means of incorporation of suitable enzymes degrading polysaccharides, a method of immunization by means of oral, nasal, rectal or vaginal delivery of particles, by inhalation to the lung, and injection into muscle or skin or underneath the skin, a method of introduction of alum adjuvant as an integral part of particles and composition of matter, method of incorporation of mucoadhesive polymers into the particles and composition of matter.
In one aspect, the present invention provides a method of making particles useful in, for example, drug delivery, is provided, comprising the steps of: providing a stream of uniformly-sized submicron or few micron drops of polyanionic polymer solution by means of a hollow ultrasonic device; collecting said droplets in a stirred reactor provided with a cationic solution; wherein the polyanionic droplets and said cationic solution react to form particles. The particles have a polyanionic core and polyanionic/polycationic complex shell (corona) with excess of the positive charge on the particle periphery. Conversely, a stream of cationic solution of the size is collected in a polyanionic solution. The particles have polycationic core and polycationic/polyanionic complex shell (corona) with the excess of negative charge on the particle periphery. Alternatively, the polyanionic and polycationic solutions are mixed together in the ratio of 1:1 to 1:4 (the same ratio of polycationic to polyanionic solutions in the converse mode) and gently stirred for 5-10 minutes. For many combinations of polymers, a spontaneous formation of particles is observed. Still alternatively, streams of uniformly sized submicron droplets of both polyanionic and polycationic solutions are reacted in a gas-phase reactor.
Yet in another aspect of the present invention, there is provided a composition of matter comprising new multicomponent systems to generate microparticles, composed of a structural (gelling) polymer and a polymer providing the mechanical strength and permeability control.
In addition, an embodiment may be where the individual components of the core polyanionic solution of polymers include concentrations of 0.01 wt-% to 0.5 wt-%. A more preferred embodiment would include a composition, where each component of the polyanions is at a concentration of 0.05 wt-% to 0.2 wt-%. In addition, the individual components of the corona cationic solution are at a concentration of 0.01 wt-% to 0.5 wt-%. In a most preferred embodiment, the polycations are at 0.05 wt-% to 0.2 wt-% and calcium chloride at 0.05 wt-% to 0.2 wt-% (and potassium chloride at 0.05 wt-% to 0.2 wt-% in case carrageenans are used as anionic polymers).
In another aspect of the present invention, there is provided a composition of matter comprising of the core polymers and cationic antigens, the latter being incorporated as an integral part of the ionically formed complex.
In addition, an embodiment may be where the individual components of the core cationic solution of polymers and inorganic salts include concentrations of 0.01 wt-% to 0.5 wt-%. A more preferred embodiment would include a composition, where each component of the polycations and of inorganic salts is at a concentration of 0.05 wt-% to 0.2 wt-%. In addition, the individual components of the corona polyanionic solution are at a concentration of 0.01 wt-% to 0.5 wt-%. In a most preferred embodiment, the polycations are at 0.05 wt-% to 0.2 wt-%.
In an additional aspect, the present invention may include the composition of matter comprising of charged polymeric surface modifiers (electrostatic stabilizers), the latter being incorporated in one step together with other polymeric components as an integral part of the complex. Similarly, a nonionic polymeric surface modifier (steric stabilizer) is integrated into the polymer structure via an entrapment. Both classes of surface modifiers are included to prevent particle aggregation upon their further processing.
In yet another aspect of the present invention, there is provided a method of direct use of the said reactor content in the case of oral, nasal, rectal, and vaginal application (vaccine), application by inhalation to the lung, and injection into muscle or skin or underneath the skin.
Another aspect of the present invention provides a composition of matter comprising of anionic polymers and anionic antigens (and plasmid DNA and antisense RNA and DNA oligonucleotide), the latter being incorporated as an integral part of the ionically formed complex.
In addition, the present invention includes a method of processing of said reactor content comprising the steps of: sedimenting or centrifuging said reactor mixture; collecting microparticles or nanoparticles as a pellet; rinsing said particles in a large excess of water, buffer, cryopreservation solution, electrostatic or steric stabilizer solution; separating said suspension by said sedimentation or centrifugation step; repeating said rinsing and separation steps; and reducing volume of the said suspension to about {fraction (1/100)}th of the initial volume.
In an additional aspect of the present invention, there is provided a method of a chemical stabilization of the washed and isolated particles comprising the steps of: reacting the particles with a crosslinking agent; rinsing said particles in a large excess of water, buffer or a cryopreservation solution, electrostatic or steric stabilizer solution; separating the particles via sedimentation or centrifugation; repeating the rinsing and separation steps; and reducing volume of the suspension.
In a most preferred embodiment, the crosslinking agent is dextran polyaldehyde, a solution of photocrosslinking polymer, or a xcex3-glutamyl transferase enzyme. The reaction conditions are selected accordingly, but within the physiological realm.
In addition, the present invention may include a method of cryoprotecting said washed particles comprising the steps of: suspending the particles in a cryoprotective solution; and lyophilization of the suspension in a suitable lyophilization apparatus. A preferred embodiment would include glycerol, sucrose, PEG, PPG, PVP, block polymers of polyoxyethylene and polyoxypropylene, water soluble derivatized celluloses and some other agents at a concentration of 1 wt-% to 10 wt-%.
In another aspect of the present invention, there is provided a method of adjustment of biodegradability of polymeric mixtures, comprising the steps of: adding a suitable amount of suitable enzyme to a polysaccharide to be degraded; breaking down a polysaccharide at physiological conditions in vivo to degradation products which can be further broken to nonharmful products in animal/human body.
A preferred embodiment would include alginate-lyase (alginase) and carrageenase for polymer matrices containing alginate or carrageenans, in quantities allowing for controlled biodegradation in the range of one week to several months.
In an additional aspect of the present invention, there is provided a method of immunization of animals by means of oral delivery, or other known routes of vaccine administration, of encapsulated antigen in the particles, wherein the particles are taken up by M-cells in Peyer""s patches of the epithelial lining of the upper intestinal tract and a build-up of secretory and systemic antibodies in blood is determined.
In still another aspect of the present invention, there is provided a method of introducing an adjuvant to potentiate an immunogenic effect. The adjuvant is preferably aluminum salt enabling to gel certain polysaccharides. The preferred embodiment could include CMC, CS and HV alginate as droplet forming anionic polymers, either individually, or in a mixture, and aluminum sulfate (or any other water soluble aluminum salt), calcium chloride and a suitable polycationic polymers as a corona forming mixture.
In yet another aspect of the present invention, there is provided a method of adding mucoadhesive polymers to the corona-forming bath to provide for sticking properties in relation to mucosal areas in animal/human body.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.